Attention has lately been focused on regeneration medicine whereby a tissue produced from a stem tissue is transplanted at a site of tissue injury, thereby implementing regeneration or functional recovery of an injured tissue or organ. It is well known that transplantation of a cell sheet, as a mimic tissue, in particular, is higher in treatment effect as compared with the case of a tissue solution in which a tissue is present as a simple substance. A human epidermal keratinocyte cell sheet is now a commercial realty, and a progress in clinical application of the cell sheet for use in treatment of severe burns and so forth has since been made. With respect to this cell sheet described as above, one of problems yet to be solved is presently establishment of a method for noninvasively evaluating the cell state of a cell sheet. The cell sheet becomes a mimic tissue via three stages including a step 1 through a step 3, after the cell sheet is disseminated with cells, described as follows. The step 1: a cell is caused to adhere to a culture surface, the step 2: the cell undergoes proliferation in a single layer throughout the culture surface to form a basal layer, and the step 3: the cell is multilayered in two layers or more, and further, each of the cells, in the two layers or higher, respectively, undergoes differentiation.
With the current state of the art, the quality of a cell sheet for use in transplantation is verified by invasive evaluation, such as observation with the use of a phase-contrast microscope during culturing, histological stain against a cell sheet for use in evaluation, produced simultaneously with the cell sheet for use in transplantation, and in the same condition as is the case with the cell sheet for use in transplantation and, and so forth. However, these methods each have a disadvantage. On one hand, the observation of a cell, using the phase-contrast microscope, is noninvasive, and can be conducted whenever necessary during the culturing, however, this observation can cope with the observation of only the surface layer of the cell sheet, but cannot evaluate a cell sheet as multilayered in the step 2 onward. Further, with respect to the evaluation by the histological stain, applied to the cell sheet for use in evaluation, it is possible to evaluate an extent of the multilayering as well as differentiation, in the step 2 onward, however, since the cell sheet is fixedly attached, this evaluation represents an invasive method, and it is therefore impossible to evaluate the cell sheet itself for use in transplantation. It can be said that establishment of a noninvasive monitoring technique capable of solving these problems will enable the cell state of a cell sheet to be directly evaluated, thereby contributing to enhancement in the quality of a regenerated tissue for use in transplantation.
A method for noninvasive cell-evaluation has thus far been described in several Patent Literature. For example, in Patent Literature 1, there has been proposed a method whereby respective cells of an embryo, an egg, a karyoplast, a stem cell, and a stem cell precursor are used as a target, and a viable force of each of the targets is determined on the basis of the concentration of an amino-acid in a medium, used as an index, thereby differentiating between a healthy embryo and an abeyant embryo. This has an object of selecting the healthy embryo to be returned to a mother body in the case of an artificial insemination. If this can be realized, it will become possible to limit the number of the embryos to be returned to the mother body to one, so that multiple pregnancy can be decreased in number. Further, in Patent Literature 2, there has been proposed a method whereby a cell sheet is used as a target, and timing of multilayering is determined on the basis of a ratio of glucose concentration in medium to ammonia concentration in medium, thereby enabling proliferation to be completed at appropriate timing.